Treatment for Cardiovascular Disease

ABSTRACT

This invention relates to a method for treating and preventing hypertension by administering a therapeutically effective amount of an agent capable of reducing uric acid levels in a patient in need of such treatment. Additionally, the scope of the invention includes a method of treating coronary heart disease by administering a therapeutically effective amount of an agent capable of reducing uric acid levels in a patient in need of such treatment.

This application claims priority from co-pending provisional applicationSer. No. 60/214,825 filed on Jun. 28, 2000.

The U.S. Government has a paid-up license in this invention and a rightin limited circumstances to require the patent owner to license otherson reasonable terms as provided for by the terms of National Instituteof Health Grant No. DK 47659.

BACKGROUND OF THE INVENTION

Uric acid is a purine metabolite that in most animals is degraded by thehepatic enzyme uricase to allantoin. However, several mutations of thegene for this enzyme occurred during early primate development with theconsequence that man and other primates have relatively higher levels ofserum uric acid [Wu, X., Muzny, D. M., Lee, C. C., and Caskey, C. T.,Two independent mutational events resulted in the loss of urate oxidaseduring hominoid evolution. J. Mol. Evol. 34:78-84 (1992)]. The adaptivebenefit of this deletion is not known nor has the modern dayconsequences of these mutations been fully understood. It has beenhypothesized that the loss of uricase provided a protective benefit toprehistoric man who was known to have a very low sodium diet [Eaton, S.B., Konner, and M., Paleolithic nutrition: A consideration of its natureand current implications. N Engl J Med 312: 283-289 (1985)] but inmodern times these mutations resulted in the development of hypertensionand other cardiovascular diseases. In most subjects, the loss of uricaseappears to be of no significance, but for the 10 to 15 percent of thegeneral population with the highest uric acid levels (>6.0 mg/dl inwomen and >6.5 mg/dl in men), there is an increased risk for thedevelopment of hypertension, atherosclerosis, and other cardiovasculardiseases. Additionally 25 to 50% of hypertensive individuals haveelevated serum uric acid, based upon the current standards 7 mg/dl[Cannon, P. J., Stason, W. B., Demartini, F. E., Sommers, S. C., andLaragh, J. H., Hyperuricemia in primary and renal hypertension. N Engl JMed 275:457-464 (1966]. This invention demonstrates for the first timemechanistic evidence that uric acid is directly related to thedevelopment of increased blood pressure.

An association between an elevated uric acid and an increased risk forcardiovascular disease was originally suggested by Haig in the late1800s. Haig postulated that uric acid crystals might precipitate in thecirculation and occlude the microvasculature [Haig, A., On uric acid andarterial tension. Br Med J 1:288-291 (1889)], thereby assuming that thedamaging effects of uric acid were related to the formation of uric acidcrystals and not to the soluble form of uric acid. Recentepidemiological studies have reported that an elevated uric acid confersan increased risk for the development of hypertension [Selby, J. V.,Friedman, G. D., and Quesenberry, C. P., Precursors of essentialhypertension: pulmonary function, heart rate, uric acid, serumcholesterol, and other serum chemistries. Am J Epidemiol 131:1017-27(1990); Jossa, F., et al. Serum uric acid and hypertension: the Olivettiheart study. J Hum Hypertens 8:677-681 (1994); and Goldstein, H. S., andManowitz, P., Relationship between serum uric acid and blood pressure inadolescents. Annals Hum Biol 20:423-431 (1993)], ischemic heart disease[Fang, J., and Alderman, M. H. Serum uric acid and cardiovascularmortality. The NHANES I Epidemiologic Follow-up Study, 1971-1992 JAMA283:2404-2410 (2000); Bengtsson, C., Lapidus, L., Stendahl, C., andWaldenström, J., Hyperuricemia and risk of cardiovasular disease andoverall death. Acta Med Scand 224:549-55 (1988); and Alderman, M. H.,Cohen, H., Madhavan, S., Kivlighn, S. Serum uric acid and cardiovascularevents in successfully treated hypertensive patients. Hypertension34:144-150 (1999).], and stroke [Lehto, S., Niskanen, L., Rönnemaa, T.,and Laakso, M., Serum uric acid is a strong predictor of stroke inpatients with non-insulin dependent diabetes mellitus. Stroke 29:635-639(1998)]. In the Worksite study an increase of 1 mg/dl of uric acidconferred the same cardiovascular risk as an increase of 10 mm Hg insystolic blood pressure or 20 mg/dl of cholesterol [Alderman, M. H.,Cohen, H., Madhavan, S., and Kivlighn, S., Serum uric acid andcardiovascular events in successfully treated hypertensive patients.Hypertension 34:144-150 (1999).]. Several studies have also reportedthat the increased mortality associated with diuretic use can beattributed to the increase in uric acid induced by these agents [Franse,L. V., Pahor, M., and Barli, M. D., Serum uric acid, it's change withdiuretic use and risk of cardiovascular events in the SystolicHypertension in the Elderly Program (SHEP). American Society ofHypertension Annual Meeting, May 1999, New York.]. Others have shownthat an increased uric acid confers increased risk for cardiovascularmortality, especially in women [Fang, J., and Alderman, M. H., Serumuric acid and cardiovascular mortality. The NHANES I EpidemiologicFollow-up Study, 1971-1992 JAMA 283:2404-2410 (2000); Bengtsson, C.,Lapidus, L., Stendahl, C., and Waldenström, J., Hyperuricemia and riskof cardiovasular disease and overall death. Acta Med Scand 224:549-55(1988); and Persky, V. W., et al. Uric acid: A risk factor for coronaryheart disease? Circulation 59:969-979 (1979)]. Despite the clinical andepidemiological evidence, some authorities do not consider an elevateduric acid to be a true cardiovascular risk factor [Vaccarino, V., andKrumholz, H. M., Risk factors for cardiovascular disease: One down, manymore to evaluate. Ann Int Med 131:62-63 (1999); and Wannamethee, S. G.,Is serum uric acid a risk factor for coronary heart disease? J HumHypertens 13:153-156 (1999)]. This is because many patients with anelevated uric acid have other well-established risk factors forcardiovascular disease, such as hypertension, renal disease, obesity,dyslipidemia, and insulin resistance [Barlow, K. A., Hyperlipidemia inprimary gout. Metabolism 17:289-299 (1968) and Grahame, R., and Stott,J. T., Clinical survey of 354 patients with gout. Ann Rheum Dis29:461-468 (1970)]. Whereas some studies have found that an elevateduric acid level is an independent risk factor after controlling for thecontribution of these other risk factors by multivariate analyses [Fang,J., and Alderman, M. H., Serum uric acid and cardiovascular mortality.The NHANES I Epidemiologic Follow-up Study, 1971-1992 JAMA 283:2404-2410(2000); Bengtsson, C., Lapidus, L., Stendahl, C., and Waldenström, J.Hyperuricemia and risk of cardiovasular disease and overall death. ActaMed Scand 224:549-55 (1988); and Persky, V. W., et al. Uric acid: A riskfactor for coronary heart disease? Circulation 59:969-979 (1979)], otherstudies including the recent Framingham analysis could not [Culleton, B.F., Larson, M. G., Kannel, W. B., and Levy, D., Serum uric acid and riskfor cardiovascular disease and death: The Framingham Study. Ann InternMed 131:7-13 (1999); Klein, R., et al. Serum uric acid: its relationshipto coronary heart disease risk factors and cardiovascular disease. EvansCounty, Ga. Arch Int Med 132:401-410 (1973); and Yano, K., Reed, D. M.,and McGee, D. L., Ten year incidence of coronary heart disease in theHonolulu Heart Program: relationship to biologic and lifestylecharacteristics. Am J Epidemiol 119:653-666 (1984).]. The lack of amechanistic pathway by which uric acid can cause cardiovascular disease,coupled with the inconclusive clinical and epidemiological data, haveleft this issue unresolved. In considering this controversy, it isimportant to note that no animal model existed to study the effects of amildly elevated uric acid.

Cyclosporine (CSA) was introduced in the 1980's as an immunosuppressant,and quickly become a first line treatment in organ transplantation aswell as in other immunologically mediated diseases [Bennett, W. M., DeMattos, A., Meyer, M. M., Andoh, T. F., and Barry, J. M., Chroniccyclosporine nephropathy. The Achille's heel of immunossupressivetherapy. Kidney Int 1996; 50:1089.]. Cyclosporine has numerous sideeffects, of which two of the most important are nephrotoxicity [Myers,B. D. and Newton, L., Cyclosporine induced chronic nephropathy: anobbliterative microvascular renal injury. J Am Soc Nephrol 1991; 2: S45,and Chapman, J. R., Harding, N. G. L., Griffiths, D., and Morris, P. J.,Reversibility of cyclosporine nephrotoxicity after three monthstreatment. Lancet 1985; 1:128.] and hyperuricemia [Gores, P. F., Fryd,D. S., Sutherland D. E. R., Najarian, J. S., and Simmons, R. L.,Hyperuricemia after renal transplantation. Am J Surg 1988; 156: 397.].As many as 50% of patients taking CSA develop hyperuricemia [Kahl, L.E., Thompson, M. E., and Griffith, B. P., Gout in the heart transplantrecipient: Physiological puzzle and therapeutic challenge. Am J Med1989; 87: 289, Najarian, J. S., Fryd, D. S., and Stransd, M., A singleinstitution, randomized, prospective trial of cyclosporine versusazathioprine-antilymphocyte globulin for immunossupression in renalallograft recipients. Ann Surg 1985; 201:142 and Sutherland, D. E. R.,Fryd, D. S., and Strand, M. H., Minnesota randomized prospective trialof cyclosporine versus azathioprine-antilymphocyte globulin forimmunossupression in renal allograft recipients. Am J Kidney Dis 1985;5:318.] and 9 to 10% develop gout [West, C., Carpenter, B. J., andHakala, T. R., The incidence of gout in renal transplant recipients. AmJ Kidney Dis 1987; 10: 369.]. The hyperuricemia from CSA is thought toresult from both a decrease in GFR [Zurcher, R. M., Bock, H. A., andThiel, G., Hyperuricemia in cyclosporine treated patients: A GFR relatedeffect. Nephrol Dial Transplant 1996; 11:153.], as well as an increasein net tubular urate reabsorption [Laine, J., and Hohnberg, C.,Mechanisms of hyperuricemia in cyclosporine-treated renal transplantedchildren. Nephron 1996; 74: 318, and Marcen, R., Gallego, N., Orofino,L. et al., Impairment of tubular secretion of urate in renal transplantpatients on cyclosporine. Nephron 1995; 70: 307.]. The most importantcomplication of CSA is nephrotoxicity, which is characterizedhistologically by striped interstitial fibrosis, tubular atrophy andarteriolar hyalinosis [Bennett, W. M., De Mattos, A., Meyer, M. M.,Andoh, T. F., and Barry, J. M., Chronic cyclosporine nephropathy. TheAchille's heel of immunossupressive therapy. Kidney Int 1996; 50:1089,Myers, B., Cyclosporine nephrotoxicity. Kidney Int 1986; 30:964, andBennett, W. M., Burdmann, E. A., Andoh, T. F., Houghton, D. C.,Lindsley, J., and Elzinga, L. W., Nephrotoxicity of immunossupressivedrugs. Nephrol Dial Transplant 1994; 9:141.]. The pathogenesis of CSAnephropathy is multifactorial but likely involves afferent arteriolarvasoconstriction with activation of the renin angiotensin pathway andinhibition of nitric oxide (NO) production [Bennett, W. M., Burdmann, E.A., Andoh, T. F., Houghton, D. C., Lindsley, J., and Elzinga, L. W.:Nephrotoxicity of immunossupressive drugs. Nephrol Dial Transplant 1994;9:141, Burdmann, E. A., Andoh, T. F., Nast, C. C., et al., Prevention ofexperimental cyclosporine induced interstitial fibrosis by losartan andenalapril. Am J Physiol 1995; 269: F491, and Pichler, R., Franceschini,N., Young, B. A. et al., Pathogenesis of cyclosporine nephropathy. Rolesof angiotensin II and osteopontin. J Am Soc Nephrol 1995; 6: 1186.].

The possibility that cyclosporine induced hyperuricemia may have a rolein either mediating or exacerbating cyclosporine nephropathy has notpreviously been considered. However, it is known that hyperuricemia isalso associated with reduced renal blood flow and increased renalvascular resistance [Hoyer, P. F., Lee, I. K., Oemar, B. S., Krohn, H.P., Offner, G., and Brodhel, J., Renal handling of uric acid undercyclosporine A treatment. Pediatr Nephrol 1988; 2:18, and Messerli, F.H., Frolich, E. D., Drelinski, C. R., Suarez, D. H., and Aristimuno, G.G., Serum uric acid in essential hypertension: an indicator of renalvascular involvement. Ann Int Med 1980; 93:817.] and those patients withlong-standing gout may develop chronic tubulointerstitial disease [Beck,L. H., Requiem for gouty nephropathy. Kidney Int 30:280-287, 1986,Emmerson, B. T., and Row, P. G., An evaluation of the pathogenesis ofthe gout kidney. Kidney Int. 1975; 8:65, and Johnson, R. J., Kivlighn,S. D., Kim, Y. G., Suga, S., and Fogo, A. B., Reapprasial of thepathogenesis and consequences of hyperuricemia in hypertension,cardiovascular disease and renal disease. Am J Kidney Dis 1999; 33:225.]. Controversy has existed, however, over whether hyperuricemia isthe cause or consequence of renal vasoconstriction andtubulointerstitial lesions [Nickeleit, V., and Mihatsh, M. J., Uric acidnephropathy and end-stage renal disease. Review of a non-disease.Nephrol Dial Transplant 1997; 12: 1832, and Yu, T., Berger, L., Dorph,D. J., and Smith, H., Renal function in gout: V-Fators influencing therenal hemodynamics. Am J Med 1979: 67:766.].

A recent report suggested that allopurinol, an inhibitor of uric acidproduction, could protect the kidney from CSA nephrotoxicity [Assis, S.M., Monteiro, J. L., and Seguro, A. C., L-arginine and allopurinolprotect against cyclosporine nephrotoxicity. Transplantation 1997;63(8): 1070.]. Thus the hypothesis that hyperuricemia might exacerbatecyclosporine nephropathy was tested. As rodents normally do not becomehyperuricemic because they have the hepatic enzyme uricase, whichdegrades uric acid to allantoin [Becker, B. F., Towards thephysiological function of uric acid. Free Rad Biol Med 1993; 14:615, andWaisman, J., Bluestone, R. and Klinemberg, J. R., A preliminary reportof nephropathy in hyperuricemic rats. Lab Invest 1974; 30:716.], ratswith cyclosporine nephropathy, in the presence and absence of theuricase inhibitor, oxonic acid were compared. This inventiondemonstrates that hyperuricemia exacerbates CSA nephropathy through acrystal independent mechanism.

Hyperuricemia, defined as serum uric acid levels >7.0 mg/dl in manand >6.0 mg/dl in women, is a common metabolic abnormality that isobserved in 4 to 6% of the population (Wyngaarden J. B. and Kelley W.N., Epidemiology of hyperuricemia and gout. In Gout and Hyperuricemia,Grune and Stratton, New York, 1976, pp 21-37). The major risksclassically attributed to hyperuricemia have been the risk of developinggout and/or uric acid renal stones. Patients with longstandinghyperuricemia and/or gout are also at risk for developing chronic renaldisease (Talbot, J. H., and Terplan, K. L., The kidney in gout. Medicine39:405-467, 1960, and Gonick, H. C., Rubini, M. D., Gleason, I. O., andSommers, S. C., The renal lesion in gout. Ann Int Med 62:667-74, 1965).Several large studies have documented that between 30 and 60% ofpatients with gout will develop renal insufficiency and up to 10% willdevelop end stage renal disease (Talbot, J. H., and Terplan, K. L., Thekidney in gout. Medicine 39:405-467, 1960; Gonick, H. C., Rubini, M. D.,Gleason, I. O., and Sommers, S. C., The renal lesion in gout. Ann IntMed 62:667-74, 1965; Yu, T., Berger, L., Dorph, D. J., and Smith, H.,Renal function in gout: V-Factors influencing the renal hemodynamics. AmJ Med 67:766-71, 1979; and Berger, L., and Yu, T., Renal Function inGout: IV. An Analysis of 524 Gouty Subjects Including long-termfollow-up studies. Am J Med 59:605-613, 1975). Renal structural changesare even more common than the functional abnormalities (Greenbaum, D.,and Ross, J. H., Renal biopsy in gout., Brit Med J 1:1502-1504, 1961),and in one study renal disease was observed in 287 of 290 patients withgout (Talbot, J. H., and Terplan, K. L., The kidney in gout. Medicine39:405-467, 1960). The renal disease, which has been termed ‘goutynephropathy’, is characterized by chronic tubulointerstitial fibrosis,often with arteriolosclerosis and glomerular sclerosis (Talbot, J. H.,and Terplan, K. L., The kidney in gout. Medicine 39:405-467, 1960). Inaddition, many biopsies show focal deposits of urate crystals,particularly in the outer medulla (Talbot, J. H., and Terplan, K. L.,The kidney in gout. Medicine 39:405-467, 1960; Gonick, H. C., Rubini, M.D., Gleason, I. O., and Sommers, S. C., The renal lesion in gout. AnnInt Med 62:667-74, 1965; and Cannon, P. J., Stason, W. B., Dematini, F.E., Sommers, S. C., and Laragh, J. H., Hyperuricemia in Primary andRenal Hypertension. New Engl J Med 275:457-464, 1966).

However, investigators have challenged if ‘gouty nephropathy’ trulyexists (Beck, L. H., Requiem for gouty nephropathy. Kidney Int30:280-287, 1986, and Nickeleit, V. and Mihatsh, M. J., Uric acidnephropathy and end-stage renal disease. Review of a non-disease.Nephrol Dial Transplant 12: 1832-38, 1997). Some studies have suggestedthat the renal functional changes could be attributed to co-existinghypertension or the consequence of aging (Yü, T., Berger, L., Dorph, D.J., and Smith, H., Renal function in gout: V-Factors influencing therenal hemodynamics. Am J Med 67:766-71, 1979, and Yü, T. and Berger, L.,Impaired Renal Function in Gout: Its Association with HypertensiveVascular Disease and Intrinsic Renal Disease. Am J Med 72:95-100, 1982).Others have noted the apparent discrepancy between the focal nature ofthe urate deposits and the diffuse interstitial disease ((Beck, L. H.,Requiem for gouty nephropathy. Kidney Int 30:280-287, 1986, andNickeleit, V. and Mihatsh, M. J., Uric acid nephropathy and end-stagerenal disease. Review of a non-disease. Nephrol Dial Transplant 12:1832-38, 1997). Furthermore, the effect of uric acid lowering agents onimproving renal function in patients with gout has been variable, withboth positive (Perez-Ruiz, F., Calabozo, M., Fernandez-Lopez, M. J.,Herrero-Beites, A., Ruiz-Lucea, E., Garcia-Erasukin, G., Duruelo, J.,and Alonso-Ruiz, A., Treatment of chronic gout in patients with renalfunction impairment. An open, randomized actively controlled study. JClin Rheumatol 1999; 5:49-55, and Perez-Ruiz F, Alonso-Ruiz A, CalabozoM, Herrero-Beites A, Garcia-Erauskin G, and Ruiz-Lucca E., Efficacy ofallopurinol and benzbromarone for the control of hyperuricemia. Apathogenic approach to the treatment of primary chronic gout. Ann RheumDis 1998; 57:545-549.) and negative (Fessel, W. J., Renal Outcomes ofGout and Hyperuricemia. Am J Med 67:74-82, 1979, and Rosenfeld, J. B.,Effect of long-term allopurinol administration on serial GFR innormotensive and hypertensive hyperuricemic subjects. Adv Exp Med Biol41B:581-596, 1974) studies reported.

A novel pathway has been demonstrated where uric acid, a purinemetabolite present in the blood, actually causes hypertension and renaldisease. It is known that markedly elevated uric acid can crystallize inthe tubules of the kidney and cause kidney failure. The inventiondisclosed herein is that mildly elevated uric acid levels can also causerenal disease and hypertension. Furthermore, it has been shown that thisaction is mediated in part by activation of the renin-angiotensin systemin the kidney and by the inhibition of nitric oxide synthases (NOS)within the kidney.

SUMMARY OF THE INVENTION

This invention relates to a method for treating and preventinghypertension by administering a therapeutically effective amount of anagent capable of reducing uric acid levels in a patient in need of suchtreatment. Additionally, the scope of the invention includes a method oftreating coronary heart disease by administering a therapeuticallyeffective amount of an agent capable of reducing uric acid levels in apatient in need of such treatment. The agent, or pharmaceuticallyacceptable salt thereof, capable of reducing uric acid levels isselected from the group consisting of gene therapy, a xanthine oxidaseinhibitor, a uricosuric agent, supplements of the uricase protein and aurate channel inhibitor or combinations thereof. Also within the scopeof the invention is a pharmaceutical composition, comprising a reninangiotensin system (RAS) inhibitor, or pharmaceutically acceptable saltthereof and the agent, or pharmaceutically acceptable salt thereofcapable of reducing uric acid levels, and a pharmaceutical carrier, or acombination therapy comprising the concomitant, simultaneous orsequential administration of the RAS inhibitor, or pharmaceuticallyacceptable salt thereof, and the agent, or pharmaceutically acceptablesalt thereof, capable of reducing uric acid levels.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1—A Model of Mild Hyperuricemia in Rats. Rats treated with oxonicacid (2%) develop mild hyperuricemia compared to controls on a normalsalt diet (FIG. 1A). Light microscopy (PAS, 50×) at 7 weeks is normal(FIG. 1B) and no urate crystals are present by DeGalantha (50×) stain of100% ethanol-fixed tissue (FIG. 1C). For comparison, we have included aDeGalantha stain in rats with acute urate nephropathy showingintratubular urate crystals (FIG. 1D). [Key: ▪, oxonic acid; Δ, control]

FIG. 2—Hyperuricemia Elevates Blood Pressure in Rats. Rats placed onoxonic acid (2%) develop a modest elevation of blood pressure after 3weeks on a normal sodium (0.26% NaCl) diet. *p<0.05 versus control.[Key: ▪, oxonic acid; Δ, control]

FIG. 3—Hyperuricemia Maintains and Elevates Blood Pressure in Rats on aLow Salt diet. Control rats placed on a mild salt restriction (0.125%NaCl diet) have a fall in blood pressure after several weeks; this isprevented in the presence of oxonic acid (2%). Rats placed on oxonicacid and low salt diet that are also administered allopurinol do notshow the increase in blood pressure. FIG. 3A shows the blood pressures,and FIG. 3B shows the uric acid levels. [Key: ▪, oxonic acid; Δ,control; ◯, allopurinol+oxonic acid]*p<0.05 versus control, †p<0.05 vs.oxonic acid alone.

FIG. 4—Hyperuricemia Correlates with Blood Pressure. Shown are the serumuric acid levels in individual rats on a low salt diet (closed circles),low salt+oxonic acid (open circles) and on a low salt diet+oxonicacid+allopurinol (triangles) at 7 weeks with the corresponding systolicblood pressures. A strong correlation is present (r=0.7, n=52, p<0.001).

FIG. 5—Effect of Allopurinol Intervention or Oxonic Acid Withdrawal onBlood Pressure in Hyperuricemic Rats. Rats were placed on low salt dietplus oxonic acid (2%) for 7 weeks and then matched on basis of uric acidlevel and blood pressure into 3 groups (Oxonic acid plus low salt diet(OA/LSD); Withdrawal of oxonic acid but continuation of the LSD (OAwithdrawal); and the addition of allopurinol (150 mg/L drinking water)with continuation of the OA/LSD diet (+allopurinol). FIG. 5A shows theblood pressures, and FIG. 5B shows the uric acid levels. [Key: ▪, oxonicacid; Δ, oxonic acid withdrawal; ◯, allopurinol+oxonic acid].

FIG. 6—Renal Fibrosis Develops in Hyperuricemic Rats. Rats treated withoxonic acid (2%) and a low salt diet for 11 weeks develop significantstriped interstitial fibrosis, as shown by immunostaining forinterstitial type III collagen (FIG. 6 A). Control rats on a low saltdiet do not develop any evidence of interstitial disease (FIG. 6B).Renal fibrosis was less in oxonic acid-treated rats in which allopurinolwas administered for 4 weeks prior to sacrifice (FIG. 6C).

FIG. 7—Renin Correlates with Serum Uric Acid in Rats on a Low Salt Diet.There was a direct correlation between renin (measured as the % ofglomeruli with juxtaglomerular renin staining) and serum uric acidlevels in rats on a low sodium diet (r=0.7, n=18, p=0.0006). [Key: ◯,oxonic acid; , low salt diet control; Δ, allopurinol+oxonic acid].

FIG. 8—The Elevated BP in Hyperuricemic Rats is Prevented by Treatmentwith an ACE inhibitor or with L-Arginine. Rats placed on a low salt dietwith oxonic acid develop an elevated blood pressure (OA/LSD), which isprevented if enalapril (1 mg/kg/d) or L-Arginine (1%) is added to thedrinking water. *p<0.05 versus OA/LSD. [Key: ▪, oxonic acid; ♦,L-arginine;

, enalapril].

FIG. 9—Hyperuricemia exacerbates chronic cyclosporine nephropathy.Cyclosporine alone results in classic chronic tubulointerstitial disease[A], which is worse in rats that are also hyperuricemic [B](stripedfibrosis indicated by arrows). Similarly, Cyclosporine-Oxonic Acid ratsshow greater osteopontin expression [D], macrophage infiltration [F] andtype HI collagen deposition [H], compared to respective controls[C,E,G]. (Magnification: hematoxilin-eosin ×50, OPN ×25, ED-1 ×100, typeIII collagen ×50).

FIG. 10—Effect of Hyperuricemia alone and in combination withcyclosporine on renal interstitium. Group 3, cyclosporine treated rats(CSA, gray columns) presented increased interstitial fibrosis score [A],arteriolar hyalinosis [B], osteopontin expression [C], macrophageinfiltration [D] and type III collagen deposition [E], compared to Group1, vehicle treated rats (VEH, white columns). These findings aregreatest in group 4, rats treated with both cyclosporine and oxonic acid(CSA-OA group, black columns). (*p<0.05 compared to VEH, **p<0.05compared to VEH and CSA).

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to a method of treating hypertension comprisingadministering a therapeutically effective amount of an agent capable ofreducing uric acid levels in a patient in need of such treatment. Areduction in uric acid levels would reduce the risk of hypertension,coronary heart disease, renal dysfunction, cardiovascular morbidity andmortality. Current standards for elevated uric acid levels are 7 mg/dl.However, patients with uric acid levels of 10 mg/dl are a high risk forthe above-noted cardiovascular conditions, between 6 and 10 mg/dl are atan increased risk for the above-noted cardiovascular conditions, or areduced risk with uric acid levels of >4 and <6 mg/dl.

A method of preventing hypertension comprising administering atherapeutically effective amount of an agent capable of reducing uricacid levels in a patient in need of such treatment.

A method of treating coronary heart disease comprising administering atherapeutically effective amount of an agent capable of reducing uricacid levels in a patient in need of such treatment.

A method of treating and preventing eclampsia comprising administering atherapeutically effective amount of an agent capable of reducing uricacid levels in a patient in need of such treatment.

An agent capable of reducing uric acid levels by about 0.2 mg/dl. Theagent capable of reducing uric acid levels, which is selected from thegroup consisting of: gene therapy, a xanthine oxidase inhibitor; auricosuric agent; supplements of the uricase protein and a urate channelinhibitor, or combinations of these agents. Specific examples of agentsthat are capable of reducing uric acid levels include but are notlimited to:

-   -   a gene therapy such as one that targets the overexpression of        uricase, the enzyme responsible for the breakdown of uric acid        to allantoin;    -   a xanthine oxidase inhibitor, such as allopurinol, and        carprofen;    -   a uricosuric agent, which is defined as an inhibitor of the        organic anion transport channels and/or voltage sensitive        transport channels acting in the kidney, such agents include but        are not limited to: losartan, benzbromaraone, benziodarone,        probenecid, sulfinpyrazone ethebencid, orotic acid, ticrynafen        and zoxazolamine;    -   a supplement of the uricase protein which might be delivered as        a conjugate with polyethylene glycol or another delivery system;        and    -   a urate channel inhibitor, is a means for interfering with the        uric acid transport mechanism by blocking the influx of uric        acid into cells.

A pharmaceutical composition comprising a renin angiotensin system (RAS)inhibitor or a pharmaceutically acceptable salt thereof and the agentcapable of reducing uric acid levels or a pharmaceutically acceptablesalt thereof as recited above and a pharmaceutical carrier. The reninangiotensin system inhibitor, such as an angiotensin converting enzymeinhibitor, an angiotensin II antagonist and a renin inhibitor.Representative RAS inhibitors include: captopril, cilazapril, enalapril,fosinopril, lisinopril, quinapril, ramapril, zofenopril, candesartancilexetil, eprosartan, irbesartan, losartan, tasosartan, tehnisartan,and valsartan, or pharmaceutically acceptable salts thereof. Also withinthe scope of this invention is the combination of an agent capable ofreducing uric acid levels with a combination RAS inhibitor with adiuretic, such as hydrochlorothiazide, furosemide, etc. Specificexamples, include but are not limited to the above RAS inhibitors withhydrochlorothiazide.

A combination therapy comprising the administration, concomitantly,simultaneously or sequentially, of therapeutically effective amounts ofa RAS inhibitor and the agent capable of reducing uric acid levels asrecited above. Also within the scope of this invention is thecombination therapy as recited above that includes an agent capable ofreducing uric acid levels with a combination RAS inhibitor with adiuretic, such as hydrochlorothiazide, furosemide, etc. Specificexamples, include but are not limited to the above RAS inhibitors withhydrochlorothiazide.

Also within the scope of the invention is a pharmaceutical compositioncomprising an agent which stimulates nitric oxide production viaendothelial and/or neuronal nitric oxide synthase or a pharmaceuticallyacceptable salt thereof and the agent capable of reducing uric acidlevels or a pharmaceutically acceptable salt thereof as recited aboveand a pharmaceutical carrier. An agent which stimulates nitric oxideproduction via endothelial and/or neuronal nitric oxide including, butnot limited to L-Arginine, nitrates and nitrate-mimetics and genetherapy, such as one that targets the overexpression endothelial and/orneuronal nitric oxide synthase.

The studies were performed on rats in which the serum uric acid wasraised to a mild (1.5 to 2.0-fold) degree, using an enzyme inhibitor ofuricase, an enzyme involved in the degradation of uric acid. Rats mademildly hyperuricemic developed significant hypertension within a fewweeks, and this was associated with stimulation of renin (documented byrenin staining in the kidney) and by a fall in neuronal NOS in themacula densa (a tubular segment in the kidney involved in regulation ofrenal blood flow) and of endothelial cell NOS. Associated with thesefindings was the development of renal fibrosis with increased collagendeposition and macrophage infiltration. These changes could be preventedby lowering the uric acid with allopurinol.

The studies provide a mechanism for the long-observed association ofuric acid with hypertension, cardiovascular disease and renal disease,and for the first time provides direct experimental evidence that uricacid is causal rather than simply a marker for associated cardiovascularrisk factors. It thus provides the first direct rationale for loweringuric acid as a means for not only preventing the development ofhypertension but also for its treatment—a substantial finding given that25% of the worlds population will become hypertensive. It is alsorelevant to a number of other diseases, including eclampsia (a diseaseafflicting pregnant women associated with hypertension, renal diseaseand an elevated uric acid but in which the latter was thought only to bea marker), to cyclosporine nephropathy (one of the complications oftransplantation in which hypertension, renal disease and an elevateduric acid are central features), to progressive renal disease, and evento aging associated hypertension and renal disease. The observation thatblacks have higher uric acid levels also provides a mechanism to explainthe reason they are more susceptible to hypertension.

The studies show that increasing the uric acid level in the rat willcause hypertension and renal disease, and that lowering it will lowerthe blood pressure and prevent the development of renal disease. So farwe have used pharmacologic agents for this purpose—such as the use ofallopurinol, losartan, or benziodarone. However, there are numerousother possible ways to lower uric acid—these could include repletinghumans with uricase (the enzyme that degrades uric acid to allantoin).Unlike most mammals, man had a series of mutations of the uricase geneearly in his evolution—we have hypothesized that during this period,when man was on a very low salt diet, that the increase in uric acidwould have helped maintain blood pressure under those conditions.Indeed, this was confirmed by our experimental studies in the rat.However, it may be now prudent to replace uricase in man as a means forpreventing the development of hypertension—this could be done by genetherapy or by supplying the uricase protein, such as by conjugation withpolyethylene glycol or other method. Future therapies might also bedirected at blocking the influx of uric acid into cells by interferingwith the uric acid transport mechanism.

The instant invention provides direct evidence that mild hyperuricemiain rats induces hypertension, as well as subtle renal injury andfibrosis, through a crystal-independent mechanism mediated by activationof the renin angiotensin system and downregulation of neuronal nitricoxide synthase in the macula densa. This observation may explain whyhyperuricemia has been found to predict the development of hypertension[Selby, J. V., Friedman, G. D., and Quesenberry, C. P., Precursors ofessential hypertension: pulmonary function, heart rate, uric acid, serumcholesterol, and other serum chemistries. Am J Epidemiol 131:1017-27(1990), Jossa, F., et al., Serum uric acid and hypertension: theOlivetti heart study. J Hum Hypertens 8:677-681 (1994), and Goldstein,H. S., and Manowitz, P., Relationship between serum uric acid and bloodpressure in adolescents. Annals Hum Biol 20:423-431 (1993)], and mayalso be relevant to the 25 to 50% of the hypertensive population who arefound to be hyperuricemic at presentation [Cannon, P. J., Stason, W. B.,Demartini, F. E., Sommers, S. C., and Laragh, J. H., Hyperuricemia inprimary and renal hypertension. N Engl J Med 275:457-464 (1966)]. Thesestudies may also provide a mechanism to explain how hyperuricemia canthwart the beneficial effects of diuretics on overall cardiovascularmortality [Franse, L. V., Pahor, M., and Barli, M. D., Serum uric acid,it's change with diuretic use and risk of cardiovascular events in theSystolic Hypertension in the Elderly Program (SHEP). American Society ofHypertension Annual Meeting, May 1999, New York.]. Furthermore, thefinding that hyperuricemia can induce renal fibrosis may provide amechanism for the development of ‘gouty nephropathy’ [Talbott, J. H.,and Terplan, K. L., The kidney in gout Medicine 39:405-50, 1960, andGonick, H. C., Rubini, M. E., Gleason, I. O., and Sommers, S. C., Therenal lesion in gout Annals Int Med 62:667-674, 1968], as it has beenhard to attribute the diffuse injury to urate crystal deposition alone[Beck, L. H., Requiem for gouty nephropathy Kidney Int 30:280-287(1986), and Nickeleit, V., and Mihatscli, M. J., Uric acid nephropathyand end-stage renal disease-Review of a nondisease. Nephrol Dial Transpl12:1832-1838 (1997)]. It also suggests a true pathogenic role for uricacid in familial hyperuricemic nephropathy, an inherited disorder inwhich hyperuricemia, renal vasoconstriction, hypertension andinterstitial renal disease develop [McBride, M. B., Simmonds, H. A.,Moro, F. Familial renal disease or familial juvenile hyperuricaemicnephropathy? J Inher Metab Dis 20:351-353 (1997)]. The documentationthat an elevated uric acid causes hypertension also helps resolve theclinical and epidemiological controversies surrounding the role of uricacid in cardiovascular disease, as multivariate analyses would not beexpected to show uric acid to be an independent risk factor whencontrolled for variables to which it is causally linked [Johnson, R. J.,and Tuttle, K., Much ado about nothing, or much to do about something:The continuing controversy on the role of uric acid in cardiovasculardisease. Hypertension 35:E10-E10 (2000)].

While the data suggests that an elevated uric acid can increase bloodpressure and induce renal disease through a mechanism that involvesactivation of the renin angiotensin system and inhibition of neuronalnitric oxide synthase, it is important to recognize that there may beadditional mechanisms by which uric acid contributes to cardiovasculardisease. Indeed, there are other studies have shown that uric acidremains an independent cardiovascular risk factor even after controllingfor hypertension and renal disease [Fang, J., and Alderman, M. H., Serumuric acid and cardiovascular mortality. The NHANES I EpidemiologicFollow-up Study, 1971-1992 JAMA 283:2404-2410 (2000), Bengtsson, C.,Lapidus, L., Stendahl, C., and Waldenström, J., Hyperuricemia and riskof cardiovasular disease and overall death. Acta Med Scand 224:549-55(1988), and Alderman, M. H., Cohen, H., Madhavan, S., and Kivlighn, S.,Serum uric acid and cardiovascular events in successfully treatedhypertensive patients. Hypertension 34:144-150 (1999)].

Finally, the observation that inhibition of uricase can prevent the fallof blood pressure under low salt conditions provides a mechanism toexplain why the mutations of the uricase gene, giving rise to anelevated uric acid, were preferentially conserved during early primatedevelopment. Indeed, studies had suggested that humans were on a verylow sodium diet (20-40 mmol/day of sodium) for the great majority(99.8%) of the last 3.5 million years, and it is only in the lastseveral thousand years that man has been on the modern day, high saltdiet [Eaton, S. B., and Konner, M., Paleolithic nutrition: Aconsideration of its nature and current implications. N Engl J Med 312:283-289 (1985)]. It is also of interest that studies of primitivesocieties have documented a low prevalence of hypertension andcardiovascular disease [Young, D. B., Lin, H., and McCabe, R. D.,Potassium's cardioprotective mechanisms. Am J Physiol 268:R825-R837(1995), and Tobian, L. Salt and hypertension. Lessons from animal modelsthat relate to human hypertension. Hypertension 17[suppl I]:I52-I58(1991)], suggesting that the current ‘epidemic’ of cardiovasculardisease and hypertension may be a consequence of modern society. Whilethis mutation may have benefited early humans, it is hypothesized thatin modern societies it plays a critical role in the pathogenesis ofhypertension and cardiovascular disease.

A major complication of chronic cyclosporine treatment is CSAnephropathy [Myers, B. D., Newton, L., Cyclosporine induced chronicnephropathy: an obbliterative microvascular renal injury. J Am SocNephrol 1991; 2: S45, and Myers, B., Cyclosporine nephrotoxicity, KidneyInt 1986; 30:964.], which is characterized by arteriolar hyalinosis andtubulointerstitial disease. The pathogenesis is considered to besecondary to intense renal vasoconstriction induced by angiotensin IIand other vasoactive substances [Bennett, W. M., De Mattos, A., Meyer,M. M., Andoh, T. F., and Barry, J. M., Chronic cyclosporine nephropathy.The Achille's heel of immunossupressive therapy. Kidney Int 1996;50:1089, Myers, B., Cyclosporine nephrotoxicity. Kidney Int 1986;30:964, and Bennett, W. M., Burdmann, E. A., Andoh, T. F., Houghton, D.C., Lindsley, J., and Elzinga, L. W., Nephrotoxicity ofimmunossupressive drugs. Nephrol Dial Transplant 1994; 9:141, andBurdmann, E. A., Andoh, T. F., and Nast, C. C., et al., Prevention ofexperimental cyclosporine induced interstitial fibrosis by losartan andenalapril. Am J Physiol 1995; 269: F491.].

Cyclosporine use is also associated with the development ofhyperuricemia, secondary to a decrease in uric acid excretion [Hoyer, P.F., Lee, I. K., Oemar, B. S., Krohn, H. P., Offner, G., and Brodhel, J.,Renal handling of uric acid under cyclosporine A treatment. PediatrNephrol 1988; 2:18, Cohen, S. L., Boner, G., Rosenfeld, J. B., et al.,The mechanism of hyperuricaemia in cyclosporine-treated renal transplantrecipients. Transplant Proc 1987; 19:1829, and Noordzij, T. C.,Leunissen, K. L. M., and Van Hoff, J. P., Renal handling of urate andthe incidence of gouty arthritis during cyclosporine and diuretic use.Transplantation 1991; 52(1): 64.]. While the risk of hyperuricemia inpatients on CSA has generally been considered only to be gout [West, C.,Carpenter, B. J., and Hakala, T. R., The incidence of gout in renaltransplant recipients. Am J Kidney Dis 1987; 10: 369.], it is ofinterest that there has been a longstanding controversy on the role ofhyperuricemia in mediating tubulointerstitial diseases. Numerous studieshave documented that patients with gout have a high prevalence oftubulointerstitial disease (“gouty nephropathy”) [Beck, L. H., Requiemfor gouty nephropathy. Kidney Int 30:280-287, 1986, Emmerson, B. T., andRow, P. G., An evaluation of the pathogenesis of the gout kidney. KidneyInt. 1975; 8:65, Gonick, H. C., Rubini, M. D., Gleason, I. O., andSommers, S. C. The renal lesion in gout. Ann Int Med 1965; 62:667, andTalbot, J. H., and Terplan, K. L., The kidney in gout. Medicine 1960;39:405. Steele TH: Hyperuricemic nephropathies. Nephron 1999; 81 (1suppl 1): 45.] and these patients also have evidence for intense renalvasoconstriction [Messerli, F. H., Frolich, E. D., Drelinski, G. R.,Suarez, D. H., and Mistimuno, G. G., Serum uric acid in essentialhypertension: an indicator of renal vascular involvement. Ann Int Med1980; 93:817.]. However, it has remained controversial as to whether thehyperuricemia per se contributes to the renal disease or whether therenal disease results from other associated risk factors such ashypertension [Nickeleit, V., and Mihatsh, M. J., Uric acid nephropathyand end-stage renal disease. Review of a non-disease. Nephrol DialTransplant 1997; 12: 1832.]. In this study we have addressed the role ofuric acid in a model of CSA nephropathy in rats and examined thehypothesis that hyperuricemia may significantly augment cyclosporinemediated renal injury.

The first finding was that CSA, independent of oxonic acid, wasassociated with an increase in serum uric acid with a tendency for areduction in fractional urate excretion. In rats receiving CSA andoxonic acid, the serum urate levels were higher but the fractional urateexcretion remained low to normal. These findings are similar to thoseobserved in humans [Hoyer, P. F., Lee, I. K., Oemar, B. S., Krohn, H.P., Offner, G., and Brodhel, J., Renal handling of uric acid undercyclosporine A treatment. Pediatr Nephrol 1988; 2:18, and Cohen, S. L.,Boner, G., Rosenfeld, J. B., et al., The mechanism of hyperuricaemia incyclosporine-treated renal transplant recipients. Transplant Proc 1987;19:1829.] and document the clinical relevance of this model.

The second important finding of this study was that hyperuricemiasignificantly exacerbated the tubulointerstitial disease and arteriolarhyalinosis induced by cyclosporine. Parameters analyzed includedosteopontin, which is a sensitive marker of tubulointerstitial injury,interstitial and glomerular macrophage accumulation and interstitialdeposition of type III collagen. Interestingly, all of these parameterswere significantly worse in rats treated with CSA and OA compared torats treated with CSA alone.

The mechanism by which hyperuricemia exacerbates renal disease is ofintense interest. An important finding in our study is that it does notinvolve intrarenal crystal deposition. Utilizing different stains foruric acid we were unable to identify crystals in these lesions.Furthermore, the pattern of tissue injury was more consistent with an“ischemic” pattern [Duncan, H., and Dixon, A. S., Gout, familialhyperuricemia and renal disease. Q J Med 1960; 29: 127.] as opposed toan “obstructive” pattern as seen with crystal induced intratubulardeposition [Waisman, J., Mwasi, L. M., Bluestone, R., and Klinemberg, J.R., Acute hyperuricemic nephropathy in rats. An electron microscopystudy. Am J Pathol 1975; 81(2): 367, and Tykarski, A., Evaluation ofrenal handling of uric acid in essential hypertension: hyperuricemiarelated to decreased urate secretion. Nephron 1991; 59:364.]. Inaddition the ability of CSA to reduce the fractional excretion of urateresulted in urinary levels lower than that associated with the acuteurate nephropathy model, in which urinary urate excretion is typicallyincreased [Bluestone, J., Waisman, J., and Klinemberg, J. R., Chronicexperimental hyperuricemia nephropathy. Biochemical and morphologicalcharacterization. Lab Invest 1975; 33(3): 273.].

Therefore, it is our contention that hyperuricemia may augment renalinjury in this model by potentiating CSA-mediated renalvasoconstriction. Furthermore, gout is associated with bothtubulointerstitial disease and renal vasoconstriction. It is of interestthat familial hyperuricemic nephropathy is characterized by reducedfractional urate excretion, renal vasoconstriction andtubulointerstitial disease in which intrarenal urate crystal depositionis often absent [Mateos, F. A., and Puig, J. C., Renal hemodynamics infamilial nephropathy associated with hyperuricemia. Adv Exp Med Biol1991; 309: 301, and Simmonds, H. A., Warren, D. J., Cameron, J. S.,Potter, C. F., and Farebrother, D. A., Familial gout and renal failurein young women. Clin Nephrol 1980; 14:176.].

The presence of hyperuricemia in rats with CSA-induced nephropathy isassociated with significantly worse tubulointerstitial renal injury, butdoes not involve intrarenal crystal deposition. This finding hassignificant implications not only in our understanding of thepathogenesis of CSA nephropathy, but also in the role of hyperuricemiain the progression of renal disease.

The following examples illustrate this method of treatment/prevention,and as such are not to be considered as limiting the invention set forthin the claims appended hereto.

EXAMPLES Experimental Design

All studies utilized adult male Sprague-Dawley rats (SimonsenLaboratories, Gilroy Calif.) (200-250 g).

Example 1

Rats were placed on a normal salt (NaCl 0.26%) diet with or without 2%oxonic acid (Ziegler Bros, Gardners, Pa.) added to the diet and ratswere sacrificed at week 7.

Systolic blood pressure was measured by tail cuff sphyngomanometer usingan automated system with photoelectric sensor (IITC, Life Science) thathas been shown to closely correlate with intra-arterial blood pressuremeasurements [Fischer E, Schnermann, J., Briggs, J. P, Kriz, W. Ronco,P. M., Bachmann, S. Ontogeny of NO synthase and renin in juxtaglomerularapparatus of rat kidneys. Am J Physiol 268:F1164-76, 1995].

Example 2

Rats were placed on a low sodium diet (NaCl, 0.125%) with or without 2%oxonic acid for 7 weeks. A third group were administered allopurinol inthe drinking water (150 mg/L) with weekly adjustments of the dosedepending on the uric acid level.

Systolic blood pressure was measured by tail cuff sphyngomanometer usingan automated system with photoelectric sensor (IITC, Life Science) thathas been shown to closely correlate with intra-arterial blood pressuremeasurements [Fischer E, Schnermann, J., Briggs, J. P, Kriz, W. Ronco,P. M., and Bachmann, S., Ontogeny of NO synthase and renin injuxtaglomerular apparatus of rat kidneys. Am J Physiol 268:F1164-76,1995].

Example 3

Rats were placed on the low sodium diet with oxonic acid for 7 weeks,and then were matched based on uric acid level and blood pressure invarious groups to either receive allopurinol, have the oxonic acidwithdrawn from the diet, or continue the oxonic acid/low salt diet. Acontrol group of six rats were placed on the low sodium diet alone for11 weeks. All of these rats were sacrificed at week 11.

Systolic blood pressure was measured by tail cuff sphyngomanometer usingan automated system with photoelectric sensor (IITC, Life Science) thathas been shown to closely correlate with intra-arterial blood pressuremeasurements [Fischer, E., Schnermann, J., Briggs, J. P, Kriz, W.,Ronco, P. M., and Bachmann, S., Ontogeny of NO synthase and renin injuxtaglomerular apparatus of rat kidneys. Am J Physiol 268:F1164-76,1995].

Example 4

Rats were placed on either low salt diet, low salt diet with 2% oxonicacid, low salt diet with oxonic acid and enalapril (1 mg/kg/d indrinking water), or low salt diet with oxonic acid and L-Arginine (1% indrinking water).

Systolic blood pressure was measured by tail cuff sphyngomanometer usingan automated system with photoelectric sensor (IITC, Life Science) thathas been shown to closely correlate with intra-arterial blood pressuremeasurements [Fischer, E., Schnermann, J., Briggs, J. P., Kriz, W.,Ronco, P. M., and Bachmann, S., Ontogeny of NO synthase and renin injuxtaglomerular apparatus of rat kidneys. Am J Physiol 268:F1164-76,1995].

Functional Data:

Serum and urine uric acid were measured by a carbonate phosphotungstatemethod [Henry, R. J., Sobel, C., and Kim, J., A modified carbonatephosphotungstate method for the determination of uric acid andcomparison with the spectophotometric uricase method. Am J Clin Pathol1957; 28:152.]. Serum blood urea nitrogen was measured by a standard kit(Sigma, St Louis, Mo.).

Renal Immunohistochemistry:

Renal biopsies were fixed in Methyl-Carnoy's, 10% formalin or 100%ethanol and embedded in paraffin. The presence of uric acid crystals wasevaluated by staining 4-μm ethanol-fixed sections with de Galantha andmodified Von Kossa stains. Kidney tissue from rats with acute uric acidnephropathy, induced with oxonic acid and uric acid administration wasused as a positive control [Stavric, B., Johnson, W. J., and Grice, H.C., Uric acid nephropathy: An experimental model, Proc. Soc. Exp. Biol.Med. 130: 512-16 (1969).]. Light microscopy was performed in 4-μmsections of Methyl-Carnoy's fixed tissue stained with periodic acidSchiff (PAS) reagent or with hematoxylin and eosin.

Methyl-Carnoy's fixed tissue sections were analyzed by indirectimmunoperoxidase [Lombardi, D., Gordon, K. L., Polinsky, P., Suga, S.,Schwartz, S. M., and Johnson, R. J. Salt sensitive hypertension developsafter transient exposure to angiotensin II. Hypertension 33:1013-1019,1999] staining with the following primary antibodies: OP199, a goatpolyclonal antibody against osteopontin (OPN) (gift of C. Giachelli,Univ of WA, Seattle); ED-1, a monoclonal antibody to rat monocytes andmacrophages (Serotec); goat antihuman type III collagen (SouthernBiotechnology Associates Inc, Birmingham Ala.); and anti-renin, a mouseantibody to human renin (Sanofi Recherche, Montpellier, France).Sections were incubated with a secondary antibody followed byhorseradish peroxidase-conjugated avidin D (Vector Laboratories,Burlingame, Calif.), diaminobenzidine (Sigma) with or without nickelchloride as a chromogen, and counsterstained with methyl green.

NOS1 was detected on formalin fixed tissue sections with a rabbitanti-rat neuronal nitric oxide synthase (Transduction Laboratories,Lexington, Ky.), followed by a biotinylated secondary antibody,diaminobenzidine with nickel chloride and counterstained with nuclearfast red.

Quantification of Morphologic Data:

AU quantification was performed blinded. The tubular expression ofosteopontin (OPN), which is a sensitive marker of tubulointerstitialinjury, was calculated as the percent of renal cortex occupied byOPN-positive tubules [Lombardi, D., Gordon, K. L., Polinsky, P., Suga,S., Schwartz, S. M., and Johnson, R. J. Salt sensitive hypertensiondevelops after transient exposure to angiotensin II. Hypertension33:1013-1019, 1999]. Utilizing computer-assisted image analysis software(Optimas V6.2, Media Cybernetics, Silver Springs, Md.) and digitizedimages, the percent of area occupied by OPN positive tubules per 4 mm²field at a magnification of 50× was measured and the mean percent areacalculated for each biopsy. The interstitial deposition of collagen typeIII was calculated as the % of renal cortex occupied by collagen III,noted by immunostaining, by computer image analysis. The mean number ofinterstitial macrophages (ED-1+ cells) in each biopsy was calculated ina blinded manner by counting the total number of positive interstitialcortical cells in 20 sequentially selected 0.25 mm² grids at 200×magnification. Renin expression was quantified by the number ofglomeruli with positive staining for juxtaglomerular renin using aminimum of 100 glomeruli in each biopsy as previously described [Eng,E., et al., Renal proliferation and phenotypic changes in rats withtwo-kidney, one-clip Goldblatt hypertension. Am J Hypertens 7:177-185(1994)]; this has been shown previously to correlate with intrarenalrenin content [Eng, E., et al., Renal proliferation and phenotypicchanges in rats with two-kidney, one-clip Goldblatt hypertension. Am JHypertens 7:177-185 (1994)]. NOS1 was quantified by a blinded countingof the number of positive macula densa cells staining with anti-NOS1antibody using a minimum of 100 glomeruli per biopsy [Eng, E., et al.,Renal proliferation and phenotypic changes in rats with two-kidney,one-clip Goldblatt hypertension. Am J Hypertens 7:177-185 (1994)].Previous studies have shown that the number of NOS1 cells correlateswith intrarenal NOS1 activity [Fischer, E., Schnermann, J., Briggs, J.P, Kriz, W., Ronco, P. M., and Bachmann, S., Ontogeny of NO synthase andrenin in juxtaglomerular apparatus of rat kidneys. Am J Physiol268:F1164-76, 1995].

Statistical Analysis

All values are expressed as mean±standard error, unless otherwisestated. Statistical significance (p<0.05) was evaluated by ANOVA andunpaired Student's t test with appropriate correction for multiplecomparisons.

An Animal Model of Mild Hyperuricemia

An animal model of mild hyperuricemia was developed using the rat.Several previous groups had reported that hyperuricemia could be inducedin rats by feeding them oxonic acid, which is a uricase inhibitor[Stavric, B., Johnson, W. J., and Grice, H. C., Uric acid nephropathy:An experimental model, Proc Soc Exp Biol Med 130:512-16 (1969)]. In moststudies uric acid supplements were also added to the diet.Unfortunately, this model usually results in a six to ten-fold increasein serum uric acid levels with marked uricosuria, resulting in acuterenal failure from obstruction of the renal tubules with urate crystals[Stavric, B., Johnson, W. J., and Grice, H. C., Uric acid nephropathy:An experimental model, Proc Soc Exp Biol Med 130:512-16 (1969)].Likewise, targeted deletion of the uricase gene in mice also results inmarked hyperuricemia, intrarenal urate crystal deposition, and renalfailure [Bradley, A., and Caskey, C. T., Hyperuricemia and uratenephropathy in urate oxidase deficient mice. Proc Natl Acad Sci USA91:742-746 (1994)]. While these latter models mimic the acute uratenephropathy syndrome observed in occasional patients with cancersfollowing chemotherapy (‘tumor lysis syndrome’) [Robinson, R. R., andYarger, W. E., Acute uric acid nephropathy Arch Int Med 17:839-840(1977)], they are inappropriate models for the mild hyperuricemiaobserved in patients with cardiovascular disease.

Hyperuricemia was induced in rats by feeding 2% oxonic acid in the diet,resulting in a mild increase in the serum uric acid level. Althoughurinary uric acid increased two-fold, it was not at a level sufficientto cause intrarenal crystal deposition (FIG. 1). Routine lightmicroscopy of the kidney revealed normal histology at 7 weeks, andspecial stains for uric acid crystals were negative (FIG. 1). Ratsadministered the oxonic diet also appeared completely healthy, althoughthe body weight at the end of the study (7 weeks) was slightly lower inthe hyperuricemic animals (367.6±17 vs. 394±18 g body weight, oxonicacid diet vs. control, p<0.05).

Hyperuricemia Induces Blood Pressure Elevation

A remarkable finding was that rats with hyperuricemia developedincreased blood pressure within 4 weeks after commencing the diet (FIG.2). Systolic blood pressures averaged 10 to 30 mm Hg higher in thehyperuricemic rats compared to controls. The observation that anelevated uric acid induced an increase in blood pressure, suggests thatit might act to help maintain blood pressure in conditions associatedwith a low salt intake, such as occurred during early hominoid evolution[Eaton, S. B., and Konner, M., Paleolithic nutrition: A consideration ofits nature and current implications. N Engl J Med 312: 283-289 (1985)].As shown in FIG. 3, control rats placed on a modest sodium restricteddiet had a fall in blood pressure within 3 weeks. In contrast,hyperuricemic rats on a low salt diet showed a significant increase inblood pressure resulting in 30 to 40 mmHg differences between groups.Blood pressures showed a direct correlation with uric acid levels inboth experiments (n=52, r=0.7, p<0.0001 for the low salt study; n=12,r=0.7, p<0.0001 for the normal salt study) (FIG. 4). An increase of 0.5mg/dl in uric acid resulted in an increase in systolic blood pressure of20 mm Hg (FIG. 4). At uric acid levels of 2 mg/dl or higher(corresponding to a 50% increase in uric acid over baseline) bloodpressures were in the hypertensive range (systolic blood pressure>140 mmHg). Interestingly, rats on an oxonic acid diet that did not develophyperuricemia did not have elevated blood pressures (FIG. 4).

To document that the elevation in blood pressure was due to thehyperuricemia and not a nonspecific effect of the oxonic acid,hyperuricemic rats were co-administered the xanthine oxidase inhibitor,allopurinol, with the oxonic acid. Allopurinol administered from theinitiation of the oxonic acid diet prevented the development ofhyperuricemia and hypertension (FIGS. 3A and B). Furthermore, inhypertensive, hyperuricemic rats, either withdrawal of the oxonic acidor adding allopurinol also resulted in a reduction in the blood pressurein association with a fall in serum uric acid values (FIGS. 5A and B).

Mild Hyperuricemia Causes Renal Fibrosis

In an attempt to understand the mechanism for the hypertensive effect ofhyperuricemia, we carefully examined the kidneys of the hyperuricemicand control animals. At 7 weeks both routine light microscopy (FIG. 1)and blood urea nitrogen levels were normal in the hyperuricemic rats.However, special immunohistochemical stains showed a striped pattern ofearly interstitial fibrosis, with increased deposition of interstitialcollagen, macrophage accumulation, and with tubular expression ofosteopontin, which is a sensitive marker of tubular injury [Lombardi,D., Gordon, K. L., Polinsky, P., Suga, S., Schwartz, S. M., and Johnson,R. J., Salt sensitive hypertension develops after transient exposure toangiotensin II. Hypertension 33:1013-1019, 1999]. The administration ofallopurinol from the time the diet was initiated prevented thedevelopment of the fibrotic changes (Table 1).

A second study was also performed in which allopurinol was added oroxonic acid withdrawn at 7 weeks and then the rats were followed for anadditional 4 weeks before they were sacrificed (FIG. 5). In this studythe hyperuricemic rats showed more pronounced renal fibrosis and astatistical increase in blood urea nitrogen (Table 1) (FIG. 6). Rats inwhich the hyperuricemia was treated by either the addition ofallopurinol or by the withdrawal of oxonic acid showed significantlyless renal fibrosis and lower blood urea nitrogen levels (Table 1).

TABLE 1 Hyperuricemic Rats Develop Renal Injury Type III ED-1 OPN BUNcollagen (%) (cells/mm2) (% increase) (mg/dl) Example 2: Renal Findingsat 7 weeks after Oxonic Acid (OA) in presence/absence of allopurinol(AP) Control 5.4 ± 0.3 18.3 ± 1.3  0.9 ± 0.06 14.4 ± 1.2 (LSD) OA + LSD 8.8 ± 1.5* 27.2 ± 1.9*  1.8 ± 0.06* 23.2 ± 2.3 OA/LSD + 6.2 ± 0.6 20.5± 0.6   1.3 ± 0.15*♯ 15.2 ± 2.3 Allopurinol Example 3. Effect of OxonicAcid (OA) withdrawal or addition of Allopurinol (AP) at 7 weeks on RenalFindings at 11 weeks. Control 7.2 ± 0.6 27.8 ± 1.4  0.7 ± 0.08 16.6 ±0.9  (LSD) OA + LSD 13.9 ± 0.6* 36.5 ± 1.4* 1.33 ± 0.14* 24.1 ± 1.7* OA8.9 ± 33.0 ± 1.6*  1.08 ± 0.06*♯ 18.8 ± 0.8♯ withdrawal 1.6*♯ OA/LSD +9.6 ± 25.2 ± 2.6♯  0.97 ± 0.03*♯ 17.7 ± 0.5♯ Allopurinol 0.3*♯Abbreviations: BUN, blood urea nitrogen; ED-1, macrophages; LSD, lowNaCl diet (0.125%); OA, oxonic acid; TI, tubulointerstitial. *p < .05vs. control. 24.1 ±1.7* ♯p < 0.05 vs LSD/OA.

Hyperuricemia Activates the Renin Angiotensin System and InhibitsIntrarenal Neuronal Nitric Oxide Synthase

The ‘striped’ fibrotic pattern of renal injury is characteristic ofchronic vasoconstriction and/or ischemia, which is of interest givenMesserli's observation that hyperuricemia in man is associated withrenal vasoconstriction [Messerli, F. H., Frohlich, E. D., Dreslinski, G.R., Suarez, D. H., and Aristimuno, G. G., Serum uric acid in essentialhypertension: An indicator of renal vascular involvement. Ann Int Med93:817-821, 1980.]. The renal expression of two important vasoactivemediators were examined in these rats (Table 2). The percentage ofglomeruli with juxtaglomerular renin staining was markedly increased inthe hyperuricemic animals, a finding that correlates with increasedrenal renin content [Eng, E., et al., Renal proliferation and phenotypicchanges in rats with two-kidney, one-clip Goldblatt hypertension. Am JHypertens 7:177-185 (1994)]. There was also a direct correlation ofserum uric acid levels with the percentage of renin-positive glomeruli,both in the studies using a low salt diet (r=0.7, n=18, p=0.0006, FIG.7) and in the study using a normal salt diet (r=0.6, n=12, p=0.05).Interestingly, Saito et al., have previously reported that uric acidlevels correlate with plasma renin activity in patients with essentialhypertension [Saito, I., et. al. Serum uric acid and therenin-angiotensin system in hypertension. J Am Geriatrics Soc26:241-2471976.].

The effect of hyperuricemia on neuronal nitric oxide synthase (NOS1)expression in the macula densa, which is involved in regulating afferentarteriolar tone and tubuloglomerular feedback were also examined. Asshown in Table 2, the number of neuronal nitric oxide synthase (NOS1)positive cells in the macula densa was decreased in hyperuricemic rats.This is particularly relevant, as chronic inhibition of NOS1 has beenreported to elevate blood pressure in rats [Ollerstam, A. Pittner, J.,Persson E. G., and Thorup, C., Increased blood pressure in rats afterlong-term inhibition of the neuronal isoform of nitric oxide synthase. JClin Invest 99:2212-2218, 1997.]. As with renin, the decrease in NOS-1positive cells was largely prevented by allopurinol treatment (Table 2).

To further document a role for these mediators, we administeredenalapril, an angiotensin converting enzyme inhibitor, or L-Arginine,which is a substrate for nitric oxide production, to the hyperuricemicrats from the outset. As shown in FIG. 8, hyperuricemic control ratshave an approximately 20 mm Hg increase in systolic blood pressure overthe 7 week dietary period. L-Arginine treatment largely prevented thisincrease. Enalapril-treated hyperuricemic rats had the lowest systolicblood pressures. At 7 weeks, the systolic blood pressures in theL-Arginine and enalapril groups averaged 25 mm Hg lower than thehyperuricemic controls (p<0.05). This suggests that the hypertension andrenal disease induced by hyperuricemia are dependent on both angiotensinII and file nitric oxide system.

TABLE 2 Hyperuricemia Induces Changes in Vasoactive Mediators NOS-1Renin (positive cells/100 (% positive JGA) glomeruli) Example 2. RenalFindings at 7 weeks after Oxonic Acid (OA) in presence/absence ofAllopurinol (AP) Control (LSD) 39.6 ± 2.0 147.2 ± 12.4  OA + LSD 60.9 ±1.5* 80.4 ± 4.3* OA/LSD + Allopurinol 44.1 ± 1.6♯ 97.4 ± 5.6* Example 3.Effect of Oxonic Acid (OA) withdrawal or addition of Allopurinol (AP) at7 weeks on Renal Findings at 11 weeks. Control (LSD) 41.0 ± 1.9 104.4 ±11.5  OA + LSD 58.4 ± 0.8* 65.6 ± 7.1* OA withdrawal 50.5 ± 2.1*♯ 83.1 ±10.8 OA/LSD + Allopurinol 44.2 ± 1.3♯ 98.6 ± 5.1♯ Abbreviations: AP,allopurinol; glom, glomeruli; LSD, low NaCl diet (0.125%); OA, oxonicacid; MD, macula densa. *p < .05 vs. control. ♯p < 0.05 vs LSD/OA.

Example 5 Animals

Studies were conducted in 20 adult male Sprague-Dawley rats (SimmonsenLaboratories, Gilroy, Calif., USA) weighing 200 to 250 grams. All ratswere fed a low salt diet (0.125% NaCl) (Zeigler Bros, Gardners, Pa.),with water ad libitum. The use of low salt diet has been shown toaccelerate the development of CSA nephropathy [13,26]. In order toinduce hyperuricemia, oxonic acid 2% was added to low salt diet. Becauserats have uricase, an hepatic enzyme which degrades uric acid toallantoin, the blockade of this enzyme by oxonic acid is necessary.

Experimental Design

After one week on a low salt diet, weight-matched rats were randomlydivided into four groups:

-   Group 1 (Vehicle (VH); n=6): These rats received a daily    subcutaneous (SC) injection of olive oil, for 7 weeks.-   Group 2 (Oxonic acid plus vehicle (OA); n=4): these rats received a    daily SC injection of olive oil, 1 mg/kg, and a supplement of 2%    oxonic acid in their chow, for 7 weeks.-   Group 3 (Cyclosporine (CSA); n=6): these rats received a daily    injection of cyclosporine 15 mg/kg, for 7 weeks.-   Group 4 (Cyclosporine plus oxonic acid (CSA-OA); n=4): these rats    received a daily injection of cyclosporine 15 mg/kg, and a    supplement of 2% oxonic acid in their chow for 7 weeks.

After 7 weeks, rats were placed in individual metabolic cages for24-hour urine collection. The following day, rats were anesthetized withxylazine and ketamine, serum was collected for creatinine and uric acidmeasurements, and both kidneys were obtained for histology evaluation.Biopsies were fixed in 10% formalin, 100% ethanol or Methyl Carnoy's.

Functional Data

Serum and urine creatinine were measured by a standard picric acidmethod (Sigma Diagnostics creatinine kit, St. Louis, Mo.). Serum andurine uric acid were measured by a modified carbonate-phosphotungstatemethod [Henry, R. J., Sobel, C., and Kim, J., A modified carbonatephosphotungstate method for the determination of uric acid andcomparison with the spectophotometric uricase method. Am J Clin Pathol1957; 28:152.]. Fractional uric acid excretion and creatinine clearancewere calculated by standard formulas. Cyclosporine levels were measuredby high performance liquid chromatography (HPLC) of whole blood.

Drugs

Cyclosporine (Novartis) was diluted in olive oil to a finalconcentration of 15 mg/ml and injected SC in a dose of 15 mg/kg of bodyweight.

Renal Morphology and Immunohistochemistry

Methyl Carnoy's fixed tissue was processed and paraffin embedded, and 4μm sections were stained with PAS reagents and hematoxilin-eosin.Alcohol-fixed tissue was processed and paraffin embedded, and 4 μmsections were stained for uric acid crystals by de Galantha's and VonKossa stains. The positive control was kidney tissue from a rat withacute uric acid nephropathy, induced with oxonic acid and uric acidadministration [Waisman, J., Bluestone, R., and Klinemberg, J. R., Apreliminary report of nephropathy in hyperuricemic rats. Lab Invest1974; 30:716, Bluestone, J., Waisman, J., Klinemberg, J. R., Chronicexperimental hyperuricemia nephropathy. Biochemical and morphologicalcharacterization. Lab Invest 1975; 33(3): 273, and Waisman, J., Mwasi,L. M., Bluestone, R., and Klinemberg, J. R., Acute hyperuricemicnephropathy in rats. An electron microscopy study. Am J Pathol 1975;81(2): 367.].

Methyl-Carnoy's fixed tissue sections were analyzed by indirectimmunoperoxidase with primary antibodies against osteopontin (OP199,gift of C. Giachelli, University of Washington, Seattle, Wash.),monocytes and macrophages, ED-1, Serotec, Oxford, UK) and collagen typeIII (Southern Biotechnology Associates Inc, Birmingham, Ala., USA).

Quantification of Morphologic Data

Interstitial fibrosis was scored semi quantitatively on biopsies stainedwith PAS and hematoxilin-eosin, using the following scoring system:zero=normal interstitium and tubules, 1=mild fibrosis with minimalthickening between the tubules, 2=moderate fibrosis with moderateinterstitial thickening between the tubules, 3=severe fibrosis withsevere interstitial thickening between the tubules.

The tubular expression of osteopontin (OPN), which is a sensitive markerof tubulointerstitial injury [Giachelli, C. M., Pichler, R., andLombardi, D., Osteopontin expression in angiotensin II-inducedtubulointerstitial nephritis. Kidney Int 1994; 45: 515, and Thomas, S.E., Lombardi, D., Giachelli, C., Bohle, A., and Johnson, R. J.,Osteopontin expression, tubulointerstitial disease and essentialhypertension. Am J Hypertens 1998; 11:954.], was calculated as thepercentage (%) of renal cortex occupied by OPN-positive tubules[Johnson, R. J., Alpers, C. E., Yoshimura, A., et al., Renal injury fromangiotensin II mediated hypertension. Hypertension 1992; 19: 464.],utilizing computer-assisted image analysis software (Optimas V6.2, MediaCybernetics, Silver Systems Md.) and digitized images. The % areaoccupied by OPN positive tubules per 4 mm² field at 50× was measured andthe mean % area calculated for each biopsy. The same method was used toquantify the interstitial expression of collagen III.

The mean number of interstitial macrophages (ED-1+ cells/mm²) in eachbiopsy was calculated in a blinded manner by counting the total numberof positive cells in 20 sequentially selected 0.25 mm² grids at 200×magnification. The number of macrophages per glomerular cross section(utilizing a minimum of 100 glomeruli per biopsy) was also determined.

Statistical Analysis

All values are expressed as mean±SD, unless otherwise stated. Thedifferences between groups were compared with unpaired Student's ttests.

TABLE 3 Body weight, renal function and uric acid levels at 7 weeks ofstudy Group 1 Group 2 Group 3 Group 4 Weight (grams) 422.2 ± 31.2  421.2± 28.8   357.2 ± 28.8^(a,b)  351.2 ± 10.7^(a,b) Serum Uric Acid (mg/dl)1.62 ± 0.31  4.08 ± 1.22^(a)  3.15 ± 0.85^(a)    5.90 ± 1.55^(a,b,c)Urinary Uric Acid (mg/day) 2.19 ± 0.66  5.06 ± 2.81^(a) 3.05 ± 1.00 4.74 ± 3.22^(a) Urate/creatinine (urine) 0.20 ± 0.08 0.35 ± 0.27 0.18 ±0.09 0.23 ± 0.13 FE urate 0.12 ± 0.06 0.11 ± 0.14 0.07 ± 0.04 0.06 ±0.04 Serum Creatinine (mg/dl) 0.94 ± 0.24 0.88 ± 0.19  1.35 ± 0.52^(b) 1.42 ± 0.32^(b) Creatnine Clearance (ml/min) 1.39 ± 0.73 1.94 ± 0.73 0.96 ± 0.43^(b)  0.79 ± 0.29^(b) Cyclosporine (ng/dl) 4560.0 ± 602.0 4765.0 ± 486.0  ^(a)p < 0.05 compared to Group 1; ^(b)p < 0.05 comparedto Group 2; ^(c)p < 0.05 compared to Group 3. FE urate = fractionalexcretion of uric acid

Uric Acid

Serum uric acid in control rats on a low salt diet was 1.6±0.3 mg/dl(Table 3). In rats receiving CSA alone, the uric acid levels wereincreased almost 2-fold, and were similar to the levels in vehicle ratsin which uricase was blocked by oxonic acid (OA). Serum uric acid levelswere highest in rats treated with CSA and oxonic acid (CSA-OA). (Table3).

Urinary uric acid excretion was increased in rats fed oxonic acid (OAalone and CSA-OA groups). CSA treated rats (CSA alone and CSA-OA) hadurate/creatinine ratios comparable to normal controls and the fractionalurate excretion tended to be lower than either vehicle or OA alonegroups (p=0.06). (Table 3).

Cyclosporine Levels

CSA was measured by HPLC in whole blood at 7 weeks. No difference in CSAlevels was observed between CSA and CSA-OA rats. (Table 3).

Renal Function

Glomerular filtration rate (GFR) evaluated by serum creatinine andcreatinine clearance, were reduced in both. CSA and CSA-OA groups, butno statistical difference was observed between CSA and CSA-OA groups(Table 3).

Histological Analysis Tubulointerstitial and Micro Vascular Changes

Rats treated with CSA for 7 weeks displayed classic histologicalfindings of chronic CSA nephropathy, with arteriolar hyalinosis, tubulardilatation and atrophy in a stripped pattern extending from medulla tocortex (FIG. 9A). Similar histological findings were observed in CSA-OArats, except that the changes were more severe, including arteriolarhyalinosis (61.8% vs 44.8%, CSA vs. CSA-OA, p<0.05) (FIG. 9B). Incontrast, no significant tubulointerstitial changes were noted by lightmicroscopy of PAS stained sections from VEH or OA alone rat kidneys.

Osteopontin Expression

Osteopontin is a macrophage-adhesive protein that is expressed bytubules in CSA nephropathy and has been shown to correlate with themacrophage infiltration, tubulointerstitial fibrosis and renal function[Pichler, R., Franceschini N., Young, B. A. et al., Pathogenesis ofcyclosporine nephropathy. Roles of angiotensin II and osteopontin. J AmSoc Nephrol 1995; 6: 1186.]. Whereas minimal osteopontin is expressed innormal (VEH) control rats, a significant increase was observed in ratstreated with CSA (FIG. 9C). The highest expression was observed inCSA-OA treated rats (FIGS. 9D and 10).

Macrophage Accumulation

The marked increase in OPN expression in CSA and CSA-OA treated rats wasassociated with accumulation of ED-1+ macrophages in the interstitium(FIG. 9E). Similar to the findings of OPN, CSA-OA had a greater numberof macrophages than CSA alone (395.6±92.5 vs. 271.9±43.4 ED-1+cells/mm², p<0.05). In addition, CSA treated rats exhibited a mildglomerular macrophage accumulation, which was more pronounced in CSA-OAtreated animals (1.8±0.5 vs. 3.5±1.7 ED-1+ cells/glomerular crosssection, p<0.05) (FIGS. 9E and 10).

Type III Collagen Deposition

In normal kidney, type III collagen was minimally present in renalcortex, with slight accumulation around interlobular arteries and veins.In rats on CSA, type III collagen was increased in both the cortex andsubcapsular area, and displayed a striped interstitial pattern similarto that observed by routine light microscopy (FIG. 9G). This generalpattern was more severe in CSA-OA treated rats (FIGS. 9H and 10).

Crystal Deposition

In order to determine if the increased tubulointerstial injury observedin CSA-OA was associated with intrarenal crystal deposition,alcohol-fixed tissue was stained for uric acid crystals using both theDe Galantha and modified Von Kossa stain. Whereas crystals could easilybe identified in positive control tissue from rats with acute uratenephropathy induced by a combination of oxonic acid and uric acid, nocrystals were present in any of the experimental groups.

Example 6 Uric Acid Excretion Activity (Excerpted from U.S. Pat. No.5,260,322, Columns 41-42)

Twenty-four (24) male adults (25 to 48 years old, 161 cm to 187 cm tall,weighing 48 kg to 85 kg) were divided into 4 groups, 6 per group.Compound No. 9 [(COZAAR (losartan potassium)] was orally administeredunder hunger in the form of capsules in Example 2, in a definite dose(25 mg, 50 mg, 100 mg or 200 mg) per person, by varying the dose in eachgroup. Further in order to examine influence of diet on uric acidexcretion increasing activity of Compound No. 9, the capsule of Example2 containing 100 mg of Compound No. 9 was orally administered at 2 weeksafter the test under hunger was completed. Concentration of uric acid inurine and blood was determined by the uricase-POD method at everydefinite period of time after the administration. The results are shownin Tables 11 through 14.

As is clear from Tables 11 through 14, the concentration of uric acid inserum decreased in 4 hours after medication dose-dependently. However, atendency that the uric acid concentration was recovered to theconcentration level prior to medication was noted 24 hours after. On theother hand, when medicated after meals, the concentration of uric acidin serum was kept as it decreased even 24 hours after.

The uric acid concentration in urine dose-dependently increased from 0to 4 hours by administering Compound No. 9 in doses of 25 mg, 50 mg and100 mg per person. In the dose of 200 mg, however, the uric acidconcentration in urine did not increase dose-dependently but was keptalmost constant. On the other hand, when medicated after meals, the uricacid concentration in urine increased in 0 to 8 hours.

The foregoing results reveal that the non-peptide type compounds havingan angiotensin II receptor-antagonizing activity in accordance with thepresent concentration in blood and increasing excretion of uric acidinto urine. Accordingly, the non-peptide type compounds having anangiotensin II receptor-antagonizing activity in accordance with thepresent invention are useful as drugs for the prevention or treatment ofhyperuricemia.

TABLE 11 Change of uric acid concentration in serum with passage of timewhen administered in hunger Dose (mg/man) Concentration of Uric Acid(mg/dl) Time (hr) 25 50 100 200 0 (when administered) 5.2 ± 0.5 6.1 ±1.4 5.9 ± 0.9 5.6 ± 0.7  4 4.8 ± 0.6 5.3 ± 1.3 4.6 ± 0.7 4.3 ± 0.9 244.6 ± 0.6 5.6 ± 1.4 5.2 ± 0.8 5.0 ± 0.9

TABLE 12 Change in uric acid concentration in serum with passage of timeafter meal Dose 100 mg/man Time (hr) Concentration of Uric Acid (mg/hr)0 (when administered) 5.8 ± 1.1  4 4.9 ± 1.0 24 4.7 ± 0.9

TABLE 13 Change of uric acid excretion in urine with passage of timewhen administered in hunger Dose (mg/man) Concentration of Uric Acid(mg/hr) Time (hr) 25 50 100 200 0-4 43.0 ± 24.5 52.8 ± 4.3  81.2 ± 15.7 78.7 ± 15.3 4-8 32.4 ± 14.7 42.8 ± 8.5 36.4 ± 7.7 25.4 ± 6.6  8-12 28.7± 13.6 39.1 ± 4.4 30.1 ± 6.8 19.6 ± 5.2 12-24 19.7 ± 9.9  22.2 ± 3.819.2 ± 4.2 13.4 ± 2.3 24-40 33.2 ± 21.9 26.6 ± 5.4 28.0 ± 7.2 21.0 ± 3.0

TABLE 14 Change in uric acid excretion in urine with passage of timeafter meal Dose 100 mg/man Time (hr) Concentration of Uric Acid (mg/hr)0-4  75.9 ± 19.0 4-8 59.0 ± 3.8  8-12 31.8 ± 4.5 12-24 19.7 ± 2.5 24-4029.5 ± 4.1

1. A method of treating hypertension comprising administering atherapeutically effective amount of an agent, or pharmaceuticallyacceptable salt thereof, capable of reducing uric acid levels in apatient in need of such treatment.
 2. A method of preventinghypertension comprising administering a therapeutically effective amountof an agent, or pharmaceutically acceptable salt thereof, capable ofreducing uric acid levels in a patient in need of such treatment.
 3. Amethod of treating coronary heart disease comprising administering atherapeutically effective amount of an agent, or pharmaceuticallyacceptable salt thereof, capable of reducing uric acid levels in apatient in need of such treatment.
 4. A method of treating andpreventing eclampsia comprising administering a therapeuticallyeffective amount of an agent, or pharmaceutically acceptable saltthereof, capable of reducing uric acid levels in a patient in need ofsuch treatment.
 5. An agent capable of reducing uric acid levelsselected from the group consisting of: gene therapy, a xanthine oxidaseinhibitor; a uricosuric agent; supplements of the uricase protein and aurate channel inhibitor, or pharmaceutically acceptable salts, orcombinations therefrom.
 6. The agent capable of reducing uric acidlevels, as recited in claim 5, which is gene therapy that targets theoverexpression of uricase.
 7. The agent capable of reducing uric acidlevels, as recited in claim 5, which is a xanthine oxidase inhibitorselected from the group consisting of: allopurinol, and carprofen, orpharmaceutically acceptable salt thereof.
 8. The agent capable ofreducing uric acid levels, as recited in claim 5, which is a uricosuricagent selected from the group consisting of: losartan, benzbromarone,benziodarone, probenecid, sulfinpyrazone, ethebencid, orotic acid,ticrynafen and zoxazolamine, or pharmaceutically acceptable saltthereof.
 9. The agent capable of reducing uric acid levels, as recitedin claim 5, which is a supplement of uricase protein that is deliveredas a conjugate with polyethylene glycol or an alternate delivery system.10. A pharmaceutical composition comprising a renin angiotensin system(RAS) inhibitor, or pharmaceutically acceptable salt thereof and theagent, or pharmaceutically acceptable salt thereof, capable of reducinguric acid levels as recited in claim 5, and a pharmaceutical carrier.11. A combination therapy comprising the administration, concomitantly,simultaneously or sequentially, of therapeutically effective amounts ofa RAS inhibitor, or pharmaceutically acceptable salt thereof, and theagent, or pharmaceutically acceptable salt thereof, capable of reducinguric acid levels as recited in claim
 5. 12. The pharmaceuticalcomposition levels as recited in claim 10, further comprising adiuretic, or pharmaceutically acceptable salt thereof.
 13. A combinationtherapy comprising the administration, concomitantly, simultaneously orsequentially, of therapeutically effective amounts of a combination of aRAS inhibitor, or pharmaceutically acceptable salt thereof with adiuretic, or pharmaceutically acceptable salt thereof and the agent, orpharmaceutically acceptable salt thereof, capable of reducing uric acidlevels as recited in claim 5.